Plants in which the expression of S-adenosylhomocysteine hydrolase gene is inhibited

ABSTRACT

The present invention discloses transgenic plants in which the expression of SAHH gene present in their genome is substantially inhibited. Such plants have excellent properties such as resistance to viruses.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to transgenic organisms having variousproperties which are obtained by substantially inhibiting the expressionof S-adenosylhomocysteine hydrolase (hereinafter referred to as "SAHH")genes with recombinant DNA techniques, as well as methods for creatingsuch organisms.

BACKGROUND ART

(Application to Plants)

The improvement of plants (introduction of desirable characters thereto)has been greatly dependent on classic breeding methods of crossing withwild species or mutants. Most of the varieties for use in the culture ofornamental plants and vegetables have been created through such effortsmade by breeders. However, breeding often does not progress at all sincea gene source does not exist. In addition, because breeding generallyrequires a long period of time even if a gene source exits, theimproving of plants with genetic engineering techniques has beenpositively tried recently. For example, the creating a variety havingresistance against viral diseases by conventional breeding methods isaccompanied with many difficulties, e.g., an appropriate gene sourcecannot be found, or crossing with a wild species is difficult. Inaddition, with conventional breeding methods it is almost impossible toachieve drastic improvements, such as the creation of a dwarf plant or adramatic increase in the number of flowers, and the like by regulatingthe subtle balance of plant hormones Recently, recombinant DNAtechniques and plant tissue cultivation techniques have advanced and ithas become possible to create virus-resistant plants and dwarf plantsusing such techniques. Major strategies so far tried are as describedbelow.

(1) Creation of Virus-Resistant Plants

1) Introduction of a Viral Coat Protein Gene

In 1986, Powel Abel et al. (Science 232, 738) created a plant into whichthe coat protein gene of tobacco mosaic virus (TMV) had been introduced,and this plant was demonstrated to be TMV-resistant. Since then, anumber of similar reports have been made throughout the world withvarious combinations of plants and viruses. However, a transgenic plantobtained by this method exhibits resistance against only one virus whosecoat protein gene has been introduced (or extremely allied speciesthereof). In addition, the degree of resistance is greatly influenced bythe inoculation concentration.

2) Use of a Satellite RNA

In some viruses, there is a low molecular weight RNA called satelliteRNA. Satellite RNA depends on the parent virus for its replication and,in many cases, inhibits the growth of the parent virus to therebyremarkably reduce the symptoms induced by the virus.

By utilizing this property, it is possible to create a virus-resistantplant. To date, plants resistant to cucumber mosaic virus (Harrisson etal, Nature 328, 799) and tobacco ringspot virus (Gerlach et al., Nature328, 802) have been created by introducing the cDNA of a satellite RNAinto plants. In China, a plant integrating the cDNA of the cucumbermosaic virus satellite RNA has already been subjected to a field test toput it for practical use (Saito et al, Theor. Appl. Genet. 83, 679).However, this method is applicable to only those viruses having asatellite RNA.

3) Use of an Antisense RNA

A total or a partial cDNA of a virus is integrated into a plant so thatit is transcribed and expressed in the antisense direction. When thisplant is infected with the target virus, it is thought that theantisense RNA transcribed and the nucleic acid of the virus form acomplex (a double-stranded RNA) to thereby inhibit the synthesis ofviral proteins. As a result, the growth of the virus is inhibited.However, viral RNA is abudantly present in cells and has a complicatedhigher structure. Thus, the formation of such a complex is notconsidered easy and the effect of this method is not as great asexpected (Cuozzo et al., Bio/Technology 6, 549). Even if resistance toviruses has been achieved, such resistance is expected only against thevirus from which the cDNA was derived (or extremely allied speciesthereof), as observed in the method using a coat protein.

4) Use of a Ribozyme

A ribozyme is an RNA having an activity of self-catalyzed cleaving. Itis possible to design a base sequence for a ribozyme so that itspecifically cleaves viral RNA when the RNA is transcribed and expressedin plant cells. Similar to an antisense RNA, a ribozyme must form acomplex with viral nucleic acid in order to produce its effect. Althoughthere have not been many successful cases, for example, Edington et al.have reported that this method was effective when targeting at tobaccomosaic virus ("Viral Gene and Plant Pathogenesis").

5) Introduction of a Non-Structural Protein Gene Recently, there havebeen reports on several viruses that a transgenic plant incorporatingthe total cDNA of a viral replication enzyme gene or the cDNA having amutation exhibits a high resistance against viruses (Golemboski et al.,Proc. Natl. Acad. USA 87, 6311; Carr et al., Virology 199, 439).However, there have been reported instances where the resistanceobtained by this method is easily overcome by a virus of a differentstrain from that of the targeted virus (Zairlin et al., Virology 201,200).

(2) Change of Plant Morphology by Varying an Endogenous CytokininConcentration

If plant morphologenesis can be artificially controlled, it is possibleto improve a plant into a desirable morphology for humans. This isespecially important for flower business. Recently, research concerningmorphologenesis in higher plants has been rapidly advancing withmolecular biological techniques.

The bacteria Agrobacterium rhizogenes which infects plants and induceshair root carries a giant plasmid called Ri plasmid. A part of thisplasmid is integrated into a plant genome. It is reported that, when thethree genes of rol A, B and C in Ri plasmid have been integratedseparately in tobacco, various morphological changes are observed(Schmulling et al., EMBO J. 7, 2621). In particular, rol C has beenfound to be a gene which increases the amount of cytokinins, a kind ofplant hormone (Estruch et al., EMBO J. 10, 2889). Transgenic plantscreated with this gene exhibit changes such as the shortening ofinternodes, the lowering of plant heights, extrusion of styles, anincrease in the number of flowers, expedited flowering time and thelike. Some enterprises have already been developing a rose variety withan increased number of flowers, a dwarf variety of prairie gentian, etc.utilizing rol C.

Recently, it has been reported that SAHH binds to cytokinins (a kind ofplant hormone) in plants (Mitsui et al., Plant Cell Physiol. 34, 1089).SAHH is an enzyme which catalyzes the following reaction.

    S-Adenosylhomocysteine (SAH)+H.sub.2 O→Adenosine+Homocysteine

Methylation in cells progresses in the presence of S-adenosylmethionine(SAM) as the methyl group donor irrespective of microorganisms, animalsand plants. SAM, after supplying the methyl group, becomes SAH.Therefore, SAHH which is an enzyme that hydroylzes SAH controls theconcentrations of SAM and SAH in living organisms. SAHH is a key enzymeplaying an important role in the methylation reaction. Since there havebeen found some other proteins which bind to cytokinins, it is not clearwhether SAHH is a receptor for cytokinins with which cytokinins directlyexert their physiological activity. However, it is presumed that SAHH isdeeply involved in the exertion of the physiological activity ofcytokinins and that an endogeneous cytokinin concentration regulates themethylation reaction in which SAHH is involved. This is still a matterof conjecture, since the above-mentioned report provides no data on theeffect resulted from the binding of SAHH to cytokinins. On the otherhand, the results of Examples of the present invention suggest that theeffect of the binding of SAHH to cytokinins is that SAHH concentrationis regulates the concentration of endogenous cytokinins, as opposed towhat is conjectured in the above article.

(Application to Animals)

Described below are materials currently used as therapeutic agents forviral diseases in order to inhibit the infection with or growth ofanimal viruses.

1) Vaccines

Vaccines are mainly preventive means to inactivate invaded viruses byutilizing antibody production by an animal's immune system against viralantigens. Recently, various vaccines have been improved making free useof rapidly advanced genetic engineering and protein engineering("Molecular Virology Promoting Life Sciences", Ishihama et al. (eds.),1992, Kyoritu Shuppan Co., Ltd.). Among viruses, however, there are anumber of them which skillfully escape from the immune system andagainst which no vaccine is effective.

2) Interferons

Inferferons are proteins induced by a viral infection. Interferons notonly act on peripheral cells to make them resistant to viruses, but alsohave divergent physiological activities. It is considered thatinterferons are one of cytokines. Although they have achieved someresults in a clinical application as therapeutics for hepatitis C inJapan, their use is limited because of their serious side effects.

3) Nucleic Acid Analogues

Viral DNA/RNA synthetases and reverse transcriptases are inhibited bynucleic acid analogues. Those nucleic acid analogues which are put intoactual use as anti-AIDS agents include dideoxythymidine (ddC),azidothymidine (AZT) and dideoxyinosine (ddI). They can be expected toexert great effects as medicines, but their side effects are also greatin view of their modes of action.

4) Others

Research and development of antiviral agents today is focused uponantisense medicines and inhibitors against transcriptional controlfactors of viruses. Out of the former, a therapeutic agent developed byISIS in the United States for treating viral diseases of the eyesinduced by cytomegalovirus and herpes virus has already been testedclinically. The characteristic of antisense medicines is that one targetis specifically aimed at. Other subjects of research include proteaseinhibitors which inhibit viral proteases.

5) SAHH Inhibitors

All of the antiviral agents so far described are targeted at viruses perse. However, for the inhibition of a virus continuously growing incells, a considerable amount of an antiviral agent is necessary and yetcounter-measures should also be taken to cope with possible mutation ofthe virus to escape from the agent or possible inactivation of the agentby the virus. On the other hand, there is an idea to inhibit viralgrowth by inhibiting enzymes in those cells which have been infectedwith the virus. One embodiment of this idea is an SAHH inhibitor, whichinduces methylation inhibition in cells by inhibiting the host cells'SAHH. As a result, the cap structure is inhibited to thereby inhibit thetranslational function of a target virus. Sufficient resistance to avirus has been confirmed in in vitro experiments at a concentration ofSAHH inhibitors at which no phytotoxicity is observed (Wolfe et al, J.Med. Chem. 34, 2521). It is reported that SAHH inhibitors areparticularly effective against (-) RNA viruses and double-strandedviruses and that they also inhibit some of (+) RNA viruses and DNAviruses. Recently, there have been reported that SAHH inhibitors arealso effective against retroviruses such as HIV.

Problem for Solution by the Invention

As so far described, recombinant DNA techniques have been utilized forcreating virus-resistant plants and dwarf plants and for treating viraldiseases. However, as described above, there have been involved a numberof problems.

It is an object of the invention to solve these problems and to provideorganisms with various advantageous properties using recombinant DNAtechniques.

Hereinbelow, the characteristics of the invention will be describedbriefly in comparison with prior art.

(Application to Plants)

(1) Creation of Virus-Resistant Plants

The resistance to viruses conferred by the present invention has acharacteristic that prior art cannot achieve, i.e., effects can beexpected against plurality of viral diseases. In the future, it will bepossible to give plants a practical resistance to viruses by minimizingthose negative influences caused by the inhibition of the expression anendogenous SAHH gene through changing the kind of a promoter,controlling the degree of antisense inhibition or the like.

(2) The Changing of Plant Morphology by Varying an Endogenous

Cytokinin Concentration

Morphological changes in plants induced by the invention (such as lossof apical bud dominance, dwarfing, extrusion of styles, immature pollen,increase in the number of flowers, expedited flowering time, suppressionof aging, rooting from stems) are considered the results of influencesupon the amount of endogenous cytokinins. These influences are similarto those of rol C described above, but the mechanism of influencing ofthe present invention is completely different from that of rol C.Furthermore, since it has been demonstrated, for example, thatresistance to a wide variety of viruses is achieved mainly by theinhibition of methylation, the invention is recognized to have by fargreater effects than those observed in rol C plants. In addition,considering that these effects are induced by antisense inhibition,there is a possibility that a completely opposite character may beinduced through expression in the sense direction. For example, thesuppression of lateral buds or the promotion of aging may be highlypossible by devising an appropriate promoter. When the present inventionis practiced by such antisense inhibition as described in Examples, theresultant transgenic plant does not produce any novel foreign proteinand, thus, it will be advantageous to obtain public acceptance.

(Application to Animals)

When a resistance to viruses is conferred to cells by such antisenseinhibition as described later in the Example of yeast, cytotoxicity, ifobserved any, is simply attributable to the specific inhibition of SAHH.On the other hand, when viral growth is suppressed by an SAHH inhibitor,there have been reported a number of cases where the inhibitor itself ismetabolized to exhibit cytotoxicity in addition to the SAHH inhibition.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a photograph of the morphology of transgenic Xanthi nctobacco. More specifically, it shows local lesions of the transgenicplant when inoculated with tobacco mosaic virus.

FIG. 2 is a photograph of the morphology of transgenic Xanthi nc tobaccoshowing the appearance thereof.

FIGS. 3A and 3B are photographs of the morphology of transgenic Xanthinc tobacco showing the appearance of the plant coming into flower.

FIG. 4 is a photograph of the morphology of transgenic Xanthi nc tobaccoshowing an individual having an abnormally large number of lateral budsand a large number of flowers.

FIGS. 5A and 5B are photographs of the morphology of transgenic Xanthinc tobacco showing a floral portion thereof.

FIGS. 6A and 6B are photographs showing the level of SAHH MRNA extractedfrom transgenic tobacco.

FIG. 7 is a graph showing endogeneous cytokinin contents in transgenictobacco plants.

FIG. 8 is an illustration of a hammer-head type ribozyme used in thepresent invention.

FIG. 9 is a photograph showing the electrophoresis of DNA extracted fromtransgenic petunia.

DISCLOSURE OF THE INVENTION

The present inventors have made extensive and intensive researches intomethods for creating novel organisms using recombinant DNA techniques.As a result, the inventors have found that an organism having variousexcellent properties can be created by inhibiting the expression of SAHHgenes present in the genomic DNA of the organism. Thus, the presentinvention has been achieved.

The invention relates to transgenic organisms in which the expression ofSAHH genes present in their genomes is substantially inhibited byrecombining a gene into their genomic genes, as well as methods forcreating such organisms.

The present invention will be described below in more detail.

As an organism to be transformed by the invention, any organism may beused as long as the expression of SAHH gene can be inhibited in theorganism. Target organisms include plants suffering great damage fromplant viruses; plants into which introduction of a specific character(such as dwarfing, male sterility or increase in the number of flowers)is desired; or animals which need counter-measures against serious viraldiseases. The invention aims at a wide range of organism species since,as described later, SAHH is present universally in organisms and yetdifferences in base sequences for SAHH genes are small among species.Specific examples of target organism species of the invention are asfollows. In animals, experimental animals such as mouse and monkey maybe enumerated. In plants, dicotyledonous plants such as tobacco, tomato,potato and rose may be enumerated as suitable plants. Monocotyledonousplants such as maize, rice and lily may also be used in the invention.Further, not only angiosperms but also gymnosperms may be targetorganisms of the invention.

Now, the gene or the property which is the characteristic of thetransgenic organism of the present invention will be described below.

The transgenic organism of the present invention is characterized inthat the expression of SAHH gene present in its genome is substantiallyinhibited. As a result of the inhibition of SAHH gene expression, thefollowing changes will occur in the transgenic organism.

(1) Inhibition of Methylation Reaction

A number of reports have shown that the SAHH inhibitor described aboveexhibits an excellent growth inhibition effect against animal viruses(Wolfe et al., J. Med. Chem. 34, 2521). Inhibition of the methylationreaction at a concentration of the inhibitor at which animal cells donot undergo phytotoxicity suppresses viral growth sufficiently. It isbelieved a cap structure, which is extremely important for translation,is directly influenced by the methylation inhibition. The presentinvention has generated this phenomenon not through an inhibition bySAHH inhibitors but through the inhibition of expression of SAHH at thegenetic level.

Thus, inhibition of methylation reaction confers animals and plantsresistance to viruses. However, it is unknown as to whether theinhibition will influence upon other outside characters of transgenicorganisms.

(2) Increase in the Amount of Endogenous Cytokinins

From the Examples of the invention, it has been confirmed that theamount of free, active cytokinins relatively increases by the inhibitionof expression of SAHH which is also a cytokinin-binding protein (FIG.7). Accordingly, it is considered that cytokinins are in an inactivatedstate through the binding to SAHH. Cytokinins regulated by theconcentration of SAHH are plant hormones and thus even an extremelysmall change in their amount is believed to give great influences uponthe morphology of a plant. Specifically, the following changes incharacter are expected.

1. Interactions with auxins (promotion of callus formation, growth ofcells by enlargement, cell division or differentiation) For example,dwarfing, promotion of rooting, extrusion of styles, immature pollen,etc.

2. Loss of apical bud dominance (increase in lateral buds) For example,increase in the number of flowers as a result of increase in flowerbuds, increase in tillering, etc.

3. Prevention of aging (inhibition of chlorophyll decay) For example,enhancement of photosynthesis ability, retention of freshness offlowers, etc.

4. Opening of stomata (promotion of transpiration, promotion ofphotosynthesis)

5. Resistance to plant viruses

With respect to the above-mentioned phenotypes of transgenic organisms,only those individuals which have acquired desirable characters may beselected from individuals having various properties.

On the other hand, character changes in transgenic animals are unknownbecause cytokinins, which are plant hormones, are not present in animalsas functional molecules and because study of SAHH inhibitors stillremains at the cellular level.

Now, the processes for creating the transgenic organism of the inventionwill be described.

The transgenic organism of the invention is created by recombining agene into the genomic gene of the organism. The term "recombining a geneinto the genomic genes" used herein is intended to include all of thegenetic engineering techniques exerted upon the genomic DNA of thetarget organism in order to substantially inhibit the expression of SAHHgene present in the genome.

Further, "to substantially inhibit the expression of SAHH gene" means toreduce such SAHH that actually functions in cells. This includes amethod of inhibiting the transcription itself of mRNA, a method oftrapping the transcribed mRNA, a method of synthesizing a protein whichbinds to SAHH, and the like. Preferable methods for substantiallyinhibiting the expression of SAHH gene are illustrated below.

(1) A method using antisense

This is a method wherein an antisense RNA against the base sequence fora target gene whose expression should be inhibited is expressed and thisantisense RNA forms a double-stranded RNA with the target mRNA tothereby trap the target mRNA.

(2) A method using plus-sense

When a target gene is introduced into the genomic DNA in the plus-sensedirection, usually the gene is over-expressed. However, on rareoccasions, a decrease in the amount of the target mRNA is observed. Thismechanism is not clear.

(3) A method using a ribozyme

A ribozyme is an RNA molecule 50 base pairs in size at the largest andhas an antisense sequence against a target gene. The difference from theabove-mentioned method using antisense is that the target mRNA iscleaved after the formation of a double-stranded RNA. Further, theribozyme binds to the subsequent target to repeat cleaving.

(4) A method using a transcriptional control factor and a promoterregion

This is a method to interfere the binding of the proteinous factorcontrolling the expression of a target gene to the DNA transcriptionalcontrol region (promoter). Concretely, the DNA sequence for the promoteris destroyed or the expression of the transcriptional control factoritself is inhibited.

(5) A method using a protein which binds to a target protein

The above-mentioned methods (1) to (4) are characterized in that theexpression of a target gene is inhibited. On the other hand, this is amethod to inactivate a target protein already expressed by expressing aprotein which binds to it.

Among these methods, the method using antisense described in (2) aboveis preferable in the present invention.

Now, the methods (1) to (5) will be described specifically.

(1) A method using antisense

An SAHH antisense RNA means an RNA complementary to the base sequencefor the mRNA transcribed from SAHH gene. This SAHH antisense RNAhybridizes to the target mRNA to form a double-stranded RNA. The mRNAtrapped is no longer translated into a protein and is digested by anuclease in cells.

An antisense RNA may be transcribed by simply linking SAHH gene to avector in the reverse direction downstream of its promoter.

The SAHH gene used in the invention has already been disclosed by thepresent applicant (Japanese Unexamined Patent Publication No. 4-258292)and the base sequence for the gene is shown in SEQ ID NO: 1. Asdescribed earlier, SAHH is distributed widely in microorganisms, animalsand plants and, particularly, it plays an important role in methylationreaction. To date, the following reports have been made on SAHH genes.

Human (Coulter-Karis et al., Ann. Hum. Genet. 53, 169), mouse (Ogawa etal., Proc. Natl. Acad. Sci. USA 84, 719), bacteria (Sgamga et al., Proc.Natl. Acad. Sci. USA 89, 6328), nematode (Genebank accession No.M64306), slime mold (Kasir et al., Biochem. Biophys. Res. Commun. 153,359), Trypanosoma (Genebank accession No. M76556), tobacco (JapaneseUnexamined Patent Publication No. 4-258292; Mitsui et al., Plant CellPhysiolo. 34, 1089), and Madagascar periwinkle (Schr¹ der et al., PlantPhysiol. 104, 1099).

Base sequences for these genes and amino acid sequences encoded therebyare very well preserved beyond species (60-70%). This is comparable tothe protein ubiquitin of which amino acid sequences are well preservedin all species of organisms.

An SAHH gene can be obtained, for example, by the method describedbelow. First, a section from example, by the method floral axis iscultured on Murashige-skoog medium (flower bud-inducing medium)containing kinetin and indoleacetic acid. Then, mRNA is extracted fromthe resultant cultured tissue according to conventional methods and acDNA library is prepared from the extracted mRNA. Subsequently, usingmRNA extracted from an untreated tobacco floral axis and the mRNAobtained from the above cultured tissue, a cDNA probe labelled with aradioisotope such as ³² P is prepared separately.

These probes and the above cDNA library are subjected to hybridization.As a result of the hybridization, those cDNA clones are selected whichdo not hybridize to the probe from untreated floral axis but hybridizeonly to the probe from the cultured tissue. E. coli Jm109 containing thethus obtained SAHH gene was deposited at the National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology (located at 1-3, Higashi 1-chome, Tsukuba City, IbaragiPref., Japan) as Tob-SAHH-1 under the accession No. FERM BP-4873 (dateof deposit: Nov. 4, 1994). Thus, it is possible to obtain the SAHH genefrom this microorganism.

A large number of expression promoters are known. They include thosewhich are strong in transcriptional power and allow expression in everycell (such as 35S, 19S, nos); those which respond to light (such asrbc); those which respond to temperature (such as hsp); those whichreact with hormones and those which react tissue-specifically. Whenexpression of the gene in a plant is intended, important points are asfollows. Which effect of the present invention is expected; at whichstage of growth the gene is expressed; and in which tissue the gene isexpressed specifically. For example, in the case of conferring aresistance to plant viruses, preferably a promoter which allowsexpression of the gene in every cell, especially preferably a powerfulpromoter such as 35S is used. In the case of conferring male sterilityto a plant in order not to give influences upon characters of otherplants, a flower-specific promoter is preferably used. For animals,animal virus-derived promoters are mainly known as powerful promoters(e.g., SV40 early, SV40 late, MMTV-LTR and SVLTR). In the case ofconferring a resistance to viruses by SAHH antisense inhibition at thecellular level, these powerful promoters are preferably used.

With respect to the gene to be inserted in the antisense direction, anygene which codes for SAHH may be used without particular limitationsince SAHH has 60 to 70% homology at the nucleic acid level beyondspecies. It is considered that the higher the degree of relatednessbetween the organism species to be transformed and the original sourceorganism of the SAHH gene to be inserted in the antisense direction is,the more intense the antisense inhibition becomes. On the contrary, itis considered that the lower the degree of relatedness between the twoorganisms, the weaker the antisense inhibition becomes. In addition, theSAHH gene to be inserted in the antisense direction is not necessarily awhole gene. Generally, it is considered that a higher degree ofantisense inhibition will be achieved when a whole gene is inserted;however, there may be cases where sufficient effects are obtained evenwhen a part of the gene has been inserted. It should be noted, however,that preferably a DNA fragment containing the AUG at the translationstart site is used.

The selection of an expression vector for transforming animals an plantsmay vary depending on whether the target gene is to be expressed at thecellular or individual level. A vector has been created which is thesame in the basic structure (gene cassette) placing the target genebetween a promoter and a terminator but which is provided with basesequences necessary for the incorporation of the target gene into thehost DNA outside the gene cassette. For example, in plant vectors, theRB (right border) and LB (left border) of a vector used in anAgrobacterium-mediated method are such base sequences. In animalvectors, the LTR (long terminal repeat) of a retrovirus vector is oneexample of such a base sequence.

The transfer of an antisense SAHH gene into a host organism does notrequire a special method. In plants, this transfer may be performed, forexample, by the leaf disk technique using Agrobacterium. In animals, thetransfer may be performed by the liposome method, electroporation,microinjection or the like. When the leaf disk technique is used, atransgenic plant can be obtained by the following procedures. Anantisense SAHH gene is inserted into an appropriate plant expressionvector, which is then transferred into Agrobacterium. Subsequently, leafdisks cut from germ-free leaf of a target plant are soaked in theculture solution of the above Agrobacterium to form calluses. Atransgenic plant can be obtained by selecting only those individualswhich have been transformed. This selection may be performed by addingan appropriate antibiotic to the medium on which calluses are to beformed and judging from the presence or absence of a resistance to theantibiotic. A method of transformation using Agrobacterium is saidapplicable to only dicotyledonous plants such as tobacco and notapplicable to monocotyledonous plants (De Cleene M. 1976; Bot. Rev.42:389-466). However, according to the procedures described in theInternational Patent Publication No. WO 94/00977 and Japanese PatentApplication No. 6-27320 filed previously by the present applicant, theabove method is also applicable to monocotyledonous plants. Briefly, itbecomes possible to transform monocotyledonous plants by soaking acultured tissue which is in the process of dedifferentiation or afterdedifferentiation in the culture solution of an Agrobacterium. Further,when the target plant is a tree, it is also possible to transform itaccording to conventional methods. In trees such as pine, poplar andeucalyptus, a method of transformation has been already established as astable technology. In other trees, it is also possible to transfer anSAHH gene by suitably arranging conditions for tissue culture anddevising a method of transformation. For tissue culture of trees,detailed description is found on pages 60-73 in "Plant Biotechnology II"(published by Tokyo Kagaku Dojin Co. Ltd.). A method of transformingpoplar using Agrobacterium is reported, for example, in Confalonieri etal., "Plant Cell Report 13" 256-261 (1994).

(2) A method using plus-sense (homologous recombination)

All or a part of an SAHH gene is integrated into a plant or animalexpression vector in the original base sequence so that it istranscribed to mRNA. The obtainment of an SAHH gene and the selection ofa promoter are as described in (1) above. The probability of achievingthe inhibition of SAHH expression according to this method is thoughtconsiderably low, but not zero. The mechanism is unclear but homologousrecombination can be considered. If this assumption is correct, the basesequence introduced is significant and the transcription from thepromoter has nothing to do with the inhibition.

Anyway, the present invention includes the destruction of SAHH genes byhomologous recombination. Now, specifically illustrated below is a caseof transforming a plant at the individual level so that a plus-senseSAHH is expressed. First, a commercial plant expression vector pBI121(Toyobo) is digested with appropriate restriction enzymes to remove GUSgene. For example, the vector is co-digested with SmaI and SstI. Then,an SAHH gene cloned at the EcoRI site of a commercial plasmid pBluescript SK+ (Toyobo) is cut out with SmaI and SstI so that the samecohesive ends are generated. At this time, it is confirmed that thecohesive end produced by SmaI comes on the '5 end side of the SAHH geneand that neither SmaI site nor SstI site exists in the SAHH gene.According to the manual of a commercial ligation kit (Takara Shuzo), theSAHH fragment is linked to the vector. Then, E. coli (such as JM109) istransformed with the vector to thereby obtain a recombinant plasmid.This plasmid is amplified by conventional methods, recovered and usedfor transforming a plant. With respect to a method of transformation,plants may be transformed by the leaf disk technique or the like andanimals by the liposome method, electroporation, microinjection and thelike, as described in (1) above.

(3) A method using a ribozyme

A ribozyme is an RNA molecule 50 base pairs in size at the largest. Itis composed of a portion containing a complementary strand sequence tobind to a target SAHH RNA, a portion of preserved sequence necessary forcleaving RNA, and a portion to form a higher structure (such as hairpintype and hammer-head type). First, a base sequence is designed so thatthe sequence will bind to the mRNA of the SAHH gene to be used (thetarget RNA) and cleave it. Then, a DNA fragment having this basesequence is synthesized. Subsequently, the DNA fragment is linked to apromoter in a plant or animal expression vector as described in (1)above and integrated into the genomic DNA of an organism to betransformed. For researches into SAHH inhibition in animals at thecellular level, ribozymes may be used as described below. DNA derivedfrom a ribozyme is cloned downstream of a promoter (such as T7, T3 andSP6) of a common transcription vector to synthesize ribozyme moleculesabundantly in an in vitro transcription system. Cells are allowed todirectly take in these molecules. It is also technically possible tochemically synthesize a ribozyme. At this time, if a ribozyme issynthesized using a nucleotide derivative which will make the ribozymenuclease-tolerant, the effect are expected to become greater.Hereinbelow, the procedures to knockout the SAHH gene in a plantindividual by this method will be described specifically. For example,when the cleaving of the mRNA of the SAHH gene with a hammer-head typeribozyme is planned, first the ribozyme is designed targeting on GUC inthe mRNA of the target SAHH gene. This method of designing is introducedin detail on pages 83-88 in "JIKKENN IGAKU (Experimental Medicine)" vol.12. Briefly, a target site is determined to that a secondary structureas shown in FIG. 8 is formed. The lengths of stem I and stem II arepreferably 7-10 bases, respectively. If the target site is the GUC atpositions 660-663 in the base sequence for the tobacco SAHH gene shownin SEQ ID NO: 1, the following ribozyme may be designed.

5'GAAACACCCUGAGUCCNNNNGGACGAAACGGUCU3' (SEQ ID NO:2) (N may be any base)

When the ribozyme has been designed, a DNA sequence containing theribozyme sequence is synthesized as two oligonucleotides of a sensestrand and an antisense strand with a DNA synthesizer. At this time, itis preferable to add to the 5' end an appropriate restriction site sincethe DNA sequence is to be integrated into a vector afterward. The DNAsynthesis may be performed with a commercial DNA synthesizer such as anABI DNA synthesizer Model 380A. These two DNAs are mixed and annealed tothereby form a double-stranded DNA. After digestion with a restrictionenzyme, the double-stranded DNA is cloned, for example, downstream of35S promoter in pBI121. The resultant recombinant plasmid is used totransform a plant. The transformation at this stage may also beperformed by the leaf disk technique using Agrobacterium or the like forplants and by the liposome method, electroporation, microinjection orthe like for animals, as described in (1) and (2) above. The ribozymedesigned in this section is one example and the cleaving of the SAHHmRNA may be carried out at a different site.

(4) A method using a transcriptional control factor and a promoterregion

A. A method using a promoter region

First, the genomic genes of a target organism are separated from eachother and the promoter region controlling the transcription of the SAHHgene is specified. In the case of tobacco, a genomic library is preparedusing λ phage vector EMBL3 (Stratagene) or the like. The library isscreened using as a probe a CDNA clone of an SAHH gene which has beenisotope-labelled with a commercial random primary labelling kit (TakaraShuzo). The method for preparing this library and the method forscreening are as described in detail in Japanese Patent Publication No.5-236964. Briefly, epithelial cells of tobacco floral axis are culturedon Murashige-Skoog medium containing kinetin, indoleacetic acid,sucrose, thiamine hydrochloride and myoinositol. Then, total RNA isextracted from the tissue section. Subsequently, poly (A) RNA iscollected using an oligo-dT column and a CDNA library is prepared fromthis poly (A) RNA. A CDNA probe is prepared separately from poly (A) RNAobtained from the above cultured tissue and from poly (A) RNA extractedfrom an untreated tobacco floral axis. Then, those clones are selectedfrom the above CDNA library which hybridize only to the probe from thecultured tissue but do not hybridize to the probe from untreated tissue.

The base sequence for the resultant genomic clone is determined byconventional methods (e.g., with a DNA sequencer manufactured by ABI)and the promoter region is specified. If this promoter region isdestroyed with the homologous recombination technique or the like, thetranscription of the SAHH gene will be completely stopped. Thehomologous recombination technique is also called gene targeting. Thisis a technique utilizing the phenomenon that, when the same basesequence as the target sequence to be destroyed has been integrated intoDNA, a recombination occurs accidentally at the time of replication ofthe DNA. Specifically, the target promoter region is incorporated in thegenomic DNA by the method described in (2) above.

B. A method using a transcriptional control factor

First, the presence of a protein which binds to the promoter region isconfirmed by the gel shift assay (gel retardation assay) or the like andthen the protein (transcription factor) is purified by columnchromatography. From the purified protein, a probe for detecting thegene coding for this protein is prepared. Then, the gene is separatedusing this probe. Finally, using the separated gene fragment, theexpression of the transcription factor protein is inhibited.

The purification of a transcription factor protein may be performed, forexample, according to the procedures described in the experimentalexamples on pages 83-109 in "Laboratory Manual: Functional Analysis ofPlant Genes" (published by Maruzen Co., Ltd.).

Briefly, first, the nuclear fraction is isolated from a tissue of thetarget plant by centrifugation. To this fraction, NaCl is added toextract nuclear proteins. The resultant mixture is centrifuged at25000×g for 30 minutes and the supernatant is recovered. Thissupernatant is dialyzed in a dialysis buffer to thereby obtain a nuclearextract. Thereafter, a DNA fragment containing the promoter region ofthe SAHH gene is recovered from a gel by electrophoresis. One end of theDNA fragment is labelled with γ-³² P !ATP and polynucleotide kinase. Theabove-mentioned nuclear extract and the labelled DNA are mixed andincubated at room temperature for 30 minutes. The reaction product issubjected to electrophoresis using 5% polyacrylamide gel. The resultantgel is transferred to a filter pater, dried and then subjected toautoradiography. If a gel shift is observed, this means the presence ofa DNA-binding protein. For the purification of this protein, varioustechniques and methods may be used. For example, salting out (such asammonium sulfate salting out) utilizing difference in solubility,fractionation with an organic solvent (such as acetone and ethanol),separation methods utilizing a molecular size (such asultracentrifugation, gel filtration), immunological methods and the likemay be used. The DNA-binding protein is partially purified by acombination of column chromatographies (e.g., ion exchange, hydrophobicor affinity chromatography and the like). The kind of columnchromatography to be used is determined by the nature of the targetprotein. It is necessary to establish the optimum conditions in advanceby conducting a small-scale preliminary experiment using a portion ofthe sample.

As a method for preparing the above-mentioned probe, there are a methodwherein the sequence which binds to the promoter protein is determinedby the DNAseI footprinting or the like, and an oligonucleotide havingthe sequence is synthetized to thereby obtain a probe; a method whereinthe above DNA-binding protein is purified and a probe is prepared fromthe amino acid sequence of the protein; and the like. The second methodmay be carried out as follows, for example. A portion of the amino acidsequence for the protein is determined with an ABI protein sequencer orthe like according to the operation manual thereof. Then, based on thisinformation, the base sequence for the gene is estimated. At the sametime, an mRNA is prepared from a tissue expressing SAHH well and a cDNAlibrary is prepared therefrom in advance. The method for preparing acDNA library is as described in Japanese Unexamined Patent PublicationNo. 4-258292. Briefly, a section from the epithelium of tobacco floralaxis is cultured on Murashige-Skoog medium containing kinetin andindoleacetic acid. Then, total RNA is extracted from the culturedtissue. Subsequently, poly (A) RNA is collected from the RNA using anoligo-dT column. From this poly (A) RNA, cDNA is synthesized and a cDNAlibrary is prepared. A portion of genes is amplified by PCR and a probeis prepared from the DNA fragment obtained, or an oligonucleotide probeis prepared. The above-mentioned library is screened using the probe. Astandard PCR is as follows. Based on the information on the amino acidsequence, base sequences for the 5' and the 3'end portions (for the 3'end sequence, a complementary strand) are estimated from amino acidcodons and a primer mixture is synthesized in such a manner thatproduces as less combinations as possible. Addition of a restrictionsite to the primers will be convenient for the subsequent cloning.Tobacco DNA (2 μg) and the two primers described above are mixed anddenatured by boiling 2 minutes or the like. To the resultant mixture, abuffer, a polymerase and so forth are added to thereby obtain a reactionsolution. Generally, this reaction solution is composed of 67 mMTris-HCl buffer (pH 8.8), 16.6 mM ammonium sulfate, 6.7 mM magnesiumchloride, 10 mM mercaptoethanol, 200 mM dNTPs, 1 μg of each primer, and5 units of Taq polymerase (Takara Shuzo). Such a reaction solution isheated with a DNA thermal cycler (Takara Shuzo) at 92° C. for 1.5minutes, then left stationary at 45° C. for 2.5 minutes and furtherreacted at 72° C. for 3 minutes. These operations make one cycle andthis is repeated 25 times. The amplified DNA fragment is recovered byagarose gel electrophoresis. Then, it is labelled with an isotope andused as a probe, as described above. On the other hand, theisotope-labelling of an oligonucleotide is performed by conventionalmethods; it can be performed easily using, for example, Takara Shuzomega label kit.

The DNAseI footprinting is a method used to identify a binding regionfor a nuclear protein. The contents of this method is described indetail in "Laboratory Manual: Functional Analysis of Plant Genes"(published by Maruzen Co., Ltd.) supra. Briefly, first, a DNA fragmentof which one of the ends is labelled with ³² P and a nuclear extract aremixed and partially digested with an appropriate amount of DNaseI. Then,a partially digested DNA sample is subjected to electrophoresis using adenatured polyacrylamide gel and autoradiography. As a result, a bandshowing a ladder-like shape by each base is obtained. Since the regionto which a protein has specifically bound is protected from the cleavageby DNaseI, the band at this region becomes weaker than the other regionsor completely disappears. By simultaneously running a sample for basesequence determination in the electrophoresis, it is possible toestimate the binding region of the protein.

A method for inhibiting the gene of transcription factor protein ispreferably selected from those described in (1) to (3) above.Particularly preferable is the antisense method described in (2) in viewof the simplicity in its technology.

(5) A method using a protein which binds to the target protein

At present, it is not clear if there exists a protein which binds toSAHH. First, SAHH is labelled, mixed with an extract from an animal orplant tissue and left stationary. Then, the presence of a bindingprotein is examined by the gel shift assay. If there exists a bindingprotein, the protein is purified and the gene thereof is obtained asdescribed in (4) above. This gene is incorporated downstream of thepromoter in an expression vector so that it is expressed in the sensedirection. The target organism is transformed with this vector. Thetransformation here may also be performed by the leaf disk techniqueusing Agrobacterium or the like in plants and by the liposome method,electroporation, microinjection or the like in animals, as describedabove. The resultant transgenic organism produces the SAHH-bindingprotein excessively to thereby inhibit the activity of SAHH. When anSAHH-binding protein has not been obtained easily, the gene of an SAHHantibody may be used. First, a mouse is immunized by injecting SAHHseveral times and then its spleen is removed. The immunization of amouse and the collection of spleen cells may be performed by knownmethods such as those described on pages 148 to 151 in "BiotechnologyExperiment Manual" (published by Sankyo Shuppan Co., Ltd.). If ahybridoma of a monoclonal antibody is obtainable, it may be used. mRNAis prepared and using, for example, a commercial kit manufactured byPharmacia (Recombinant Phage Antibody System), the gene of a recombinantantibody is obtained. According to the manual, the target gene can beeasily obtained. This gene is incorporated downstream of the promoter inan expression vector in the sense direction so that the gene isoverexpressed in cells. The target organism is transformed with thisvector. The transformation here may be performed by a method similar tothose described above.

Best Modes for Carrying Out the Invention

The present invention will be described more specifically below withreference to Examples, which should not be construed as limiting thescope of the present invention.

EXAMPLE 1! Creation of Transgenic Tobacco

1! Summary of the Experimental Results

An SAHH gene isolated from tobacco was linked to a plant expressionvector downstream of its 35S promoter so that the gene is expressed inthe antisense direction in every cell. This recombinant DNA wasintroduced into the genome of tobacco by the transformation method usingAgrobacterium. More than 100 individuals of the resultant transgenictobacco were analyzed. Further, in order to confirm that each of thecharacters of the transgenic tobacco are inherited to subsequentgenerations, those individuals which were obtained from R1 seedsproduced by selfing or artificial crossing. Major changes in characterobserved are described below. Most of these changes were as expectedfrom the principle described earlier in the section titled "Means tosolve the Problem".

(1) Resistance to a plurality of viruses (FIG. 1, Tables 3 and 4),

(2) Dwarf plants (FIGS. 2 and 3), (3) Increase in the number of lateralbuds (FIGS. 2 and 4), (4) Increase in the number of flowers (Table 1),(5) Flowering time is expedited (7-10 days), (6) Inhibition of aging(FIGS. 2 and 3), (7) Male sterility (FIG. 5), (8) Change in flower color(white-red).

The finding that the expression inhibition of SAHH gene by antisenseinhibition is associated with resistance to plant viruses was madereceiving a hint from the fact that SAHH inhibitors positively studiedin the field of animal viruses toward the use as antiviral agents. Thepresent invention of which the effect has been proved in plant virusesis believed easily applicable to animal viruses. Therefore, theexpression inhibition of SAHH at the genetic level according to theinvention is not limited to plants.

Experimental methods and procedures will be described below for eachitem.

2! Preparation of a CDNA Library

A section from the floral axis epithelium of tobacco (Nicotiana tabacumBY-4) was cultured on Murashige-Skoog agar medium containing kinetin (1μM) and indoleacetic acid (1 μM) for one day. Ten grams of the resultantcultured tissue was crushed in 20 ml of an extraction buffer (4Mguanidine thiocyanate, 5 mM sodium citrate, 0.5% Sarkosyl, 2 mMβ-mercaptoethanol) with a Polytron homogenizer. The resultant solutionwas centrifuged at 4000×g for 20 minutes and the supernatant wasrecovered.

This supernatant was over-layered upon 4 ml of 5.7M cesium chloridesolution placed in a centrifuge tube and centrifuged at 28000 rpm for 20hours. Then, the supernatant was discarded and the precipitate wasrecovered. This precipitate was dissolved in 1 ml of a buffer (10 mMTris-HCl, 1 mM EDTA, 0.1% SDS). To the resultant solution, an equalvolume of a mixture containing phenol:chloroform: isoamyl alcohol(25:24:1) was added, mixed well and centrifuged to thereby recover theaqueous phase of the upper layer. To the resultant aqueous phase, 5Msodium chloride was added to give a concentration of 0.25M. Further, 2.5volumes of ethanol was added thereto and left stationary at -20° C.overnight. Then, the solution was centrifuged at 10,000×g for 20 minutesand the precipitate obtained was washed with 70% ethanol and dried underreduced pressure.

The resultant dried, standard product was dissolved in 500 ul of TEbuffer (10 mM Tris-HCl, 1 mM EDTA) to thereby obtain a solution of thetotal RNA. This RNA solution was treated at 65° C. for 5 minutes andthen quickly cooled on ice. To this solution, sodium chloride was addedto give a concentration of 0.5M and poured into an oligo-dT cellulosecolumn pre-equilibrated with TE buffer. Then, the column was washed withabout 10 volumes of a buffer (0.5M NaCl, 10 mM Tris-HCl, 1 mM EDTA).Thereafter, poly(A)⁺ RNA was eluted with TE buffer.

To the resultant eluate, 1/10 volume of 5M sodium chloride solution and2.5 volumes of ethanol were added, mixed and left stationary at -70° C.Then, the solution was centrifuged at 10,000×g.

The resultant precipitate was washed with 70% ethanol and dried, tothereby obtain 10 ug of poly(A)⁺ RNA. This poly(A)⁺ RNA was dissolved in10 ul of water and 2 ul of the resultant solution was used to prepare acDNA library. The preparation of this cDNA library was carried out usingλgt11 cDNA synthesis system and a cDNA cloning system both manufacturedby Amersham and according to the manufacturer's protocols.

3! The Screening of the Library

Two micrograms of the poly(A)⁺ RNA obtained from the above-describedcultured tissue and 10 ul of α-³² P! dCTP (3000 Ci/mmol, 10 μCi/ul) werereacted at 37° C. for one hour in 30 ul of a reaction solution 50 mMTris-HCl (pH 7.6), 2 mM DTT, 5 mM MgCl₂, 40 mM KCl, 1 mM dGTP, dATP anddTTP, 5 uM dCTP, 20 units of human placenta RNAse inhibitor, 2 ug ofoligo-dT primer, 40 units of reverse transcriptase!, to thereby preparea cDNA probe.

In a similar manner, a cDNA probe was prepared from poly(A)⁺ RNAextracted from an untreated floral axis.

Subsequently, the phage which was constituting the above cDNA librarywas allowed to infect E. coli and to propagate on LB agar medium. DNAfrom about 1400 phages was transferred to two nylon membranesseparately.

The nylon membrane to which the phage DNA had been transferred and theCDNA probe prepared above were hybridized in a solution containing 6×SSC(0.9M sodium chloride, 0.09M sodium citrate), 0.1% SDS, 5×Denhart'ssolution (0.1% Ficoll, 0.1% polyvinyl pyrrolidone, 0. 1% bovine serumalbumin) and 50 ug/ml of denatured salmon sperm DNA at 65° C. for 20hours. Then, the membrane was taken out and washed with a solutioncontaining 2×SSC and 0.5% SDS at 65° C. for one hour. After thismembrane was dried, an X ray film was adhered thereto and exposed.

As a result, 10 clones could be selected which only hybridized to theprobe from the cultured tissue but did not hybridize to the probe fromthe untreated floral axis. One of these clones was closely analyzed andit was found that the insert of this clone was small about 300 basepairs in size. Then, in order to obtain a clone having a longer insert,the cDNA library was further screened using the insert of this clone asa probe.

As a result of this screening, a clone having an insert of about 1.8 Kbase pairs. The insert of this clone was sub-cloned into a plasmidvector (Bluescript manufactured by Stratagene) and its base sequence wasdetermined by the dideoxy method. The base sequence for the insert thusdecided is shown as SEQ ID NO: 1. The length of this insert is 1812 basepairs.

4! The Cloning of a Gene into a Plant Expression Vector andTransformation of Plants

Tobacco SAHH gene was inserted into a plant expression vector, pBI121(Toyobo), at the SmaI-SacI site so that it is expressed in the antisensedirection. Using the recombinant plasmid obtained, two varieties oftobacco (Nicotiana tabacum L. cv. BY-4 and N. tabacum L. cv. Xanthi nc)were transformed by the leaf disk technique using Agrobacterium.(Hereinafter, the transgenic BY-4 tobacco and the transgenic Xanthi nctobacco are sometimes abbreviated to "SHB" and "SHX", respectively.) Inthis Example, the method described on pages 164-165 in "PlantBiotechnology II" (Yasuyuki Yamada & Yoshimi Okada (eds.), 1991, TokyoKagaku Dojin Co., Ltd.) was employed. Briefly, tobacco leaf disks weresterilized and soaked in a suspension of Agrobacterium tumefaciens(LBA4404) which had been allowed to acquire the target plasmid by thefreeze-thawing method for about 30 seconds to thereby inoculate thebacterium. The leaf disks were infected with the bacterium by culturingfor about two days in a medium containing no antibiotics. Then, the leafdisks were washed and placed on a medium containing antibiotics tothereby select transformants. Transformed calluses were selected withMurashige-Skoog medium containing kanamycin (100 μg/ml) and claforan(250 μg/ml). The transformants were bred at 25° C. under 16 hourlighting. Those which exhibited shooting were transferred to a rootingmedium to induce rooting. Thereafter, the individuals were potted. As arooting medium, Murashige-Skoog medium was used. In most of the pottedindividuals, lateral buds were formed so vigorously that herbaceouscutting of clone individuals could be easily performed. Further, most ofthose individuals were dwarfed, showing shorter internodes compared tonon-transgenic individuals. This tendency was particularly strong inBY-4. There were observed several transgenic BY-individuals which hadalmost no stem and came into flower, remaining in a rossette-like shape.Generally, flowering was seen about one week earlier and the number offlowers increased two to three times. Flowers of various colors wereobserved including those of light pink (the original color of tobaccoflower), red with uncolored spots and dark red.

Hereinbelow, the characteristics of those transgenic tobacco plants inappearance will be described below with reference to figures and tables.

FIG. 2 shows the appearance of transgenic Xanthi nc tobacco beforeflowering. The plants at the center and at the left are transgenicplants. They have more lateral buds and darker green leaves compared tothe non-transgenic plant shown at the right.

FIG. 3 shows the appearances of a transgenic Xanthi nc tobacco plant(FIG. 3A) and a transgenic BY-4 tobacco plant (FIG. 3B) both flowering.In both photographs, the plant at the right exhibiting shorterinternodes and dwarfing is a transgenic plant.

FIG. 4 shows a transgenic Xanthi nc tobacco plant having an abnormallylarge number of lateral buds and a number of flowers.

FIGS. 5A and 5B show portions of a flower of a transgenic Xanthi nctobacco plant. In about one third of the transgenic individuals,extrusion of the stigma from the flower (male sterility) was observed.The pollen of these individuals is immature and extremely low ingermination ability.

Table 1 shows the number of flowers in transgenic Xanthi nc tobaccoplants. The figures shown in the table are mean values for 10individuals measured. Since one third of the transgenic individuals donot have fertility as described above, such individuals were excludedfrom the counting of the number of seed capsules.

                  TABLE 1    ______________________________________    The Number of Flowers and the Number of Seed Capsules    in Transgenic Individuals    Plant     Number of Flowers                           Number of Seed Capsules    ______________________________________    SHX (R.sub.0)              31.1 ± 6.7                           25.1 ± 4.5    Control   17.2 ± 4.5                           13.5 ± 2.4    ______________________________________

Table 2 shows a summary of detailed observation of 20 individuals eachof transgenic tobacco plants BY-4 and Xanthi nc on the degree ofdwarfing, the number of lateral buds and the degree of stigma extrusion.

                  TABLE 2    ______________________________________    Phenotypes of Transgenic Tobacco Plants                               Xanthi nc                                        BY-4    Index         Phenotype             (N = 20) (N = 20)    ______________________________________         Stigma extrusion    1    Style has the same length as that of         stamens or is shorter than stamens.                               13       9    2    Style is somewhat longer than stamens.                               2        3    3    Style is clearly longer than stamens.                               3        5    4    Stigma is extruding from petals.                               2        2         Average index:        1.7      2.1         Dwarfing    1    Height is the same as that of non-         transgenic individuals.                               4        3    2    Somewhat dwarfed.     14       8    3    Height is less than one half of that of                               2        5         non-transgenic individuals.    4    Height is less than 1/10 of that of non-                               0        4         transgenic individuals.         Average index:        1.9      2.5         Lateral buds    1    The number of lateral buds is the same as                               0        0         that of non-transgenic individuals.    2    Formation of non-elongating lateral buds.                               3        3    3    More lateral buds than seen in non-         transgenic individuals                               15       12    4    Witches' broom        2        5         Average index:        3.0      3.1    ______________________________________

EXAMPLE 2! Creation of Transgenic Petunia

1! The Cloning of a Gene into an Expression Vector and Transformation ofPlants

In a manner similar to that described in Example 1, Tobacco SAHH genewas inserted into a plant expression vector pBI121 so that the gene isexpressed in the antisense direction. Using the resultant recombinantplasmid, petunia (varieties: H1 and No. 22) was transformed. Like inExample 1, the transformation was carried out by the leaf disktechnique. During this process, acetosyringone was added to the mediumfor coculture to give a concentration of 100 μm in order to promote theinfection of petunia leaves with Agrobacterium. The selection andredifferentiation of transformants and the like were also carried out ina manner similar to that described in Example 1. However, the saltconcentration of the rooting medium was reduced to 1/2 of the saltconcentration of Murashige-Skoog medium.

2! Detection of a DNA Fragment having the 35S Promoter Sequence

To confirm that the target DNA was integrated into the resultanttransgenic petunia, the 35S promoter region was amplified by PCR. Thesequences for the primers and the conditions employed were as follows.

3' end primer: GGATAGTGGGATTGTGCGTC (SEQ ID NO:3), 5' end primer:GGATCTAACAGAACTCGCCG (SEQ ID NO:4). One cycle consisted of three stepsof at 94° C. for 2 minutes, at 65° C. for 30 seconds and at 72° C. for 2minutes. This cycle was repeated 25 times.

The DNA fragments amplified by the above procedure were subjected toelectrophoresis. FIG. 9 shows the migration patterns of the fragments.The arrow in this Figure indicates DNA fragments containing the 35Spromoter sequence. As shown in FIG. 9, the DNA fragment is detected inH1-DH and 22-DH which are transgenic plants, but not detected in H1-Cand 22-C which are non-transgenic plants. Accordingly, transformation ofa plant can be confirmed by detecting this DNA fragment.

EXAMPLE 3! Virus Inoculation Tests on Transgenic Tobacco

Transgenic tobacco plants were bred for about two to three weeks at roomtemperature (24-27° C.) after potting and then subjected to a virusinoculation test. Carborundum was sprinkled over tobacco leaves andpurified viruses diluted with phosphate buffer were inoculated theretoat a concentration of 1-5 μg/ml by smearing. Then, the leaves wererinsed with water.

When tobacco mosaic virus (TMV) was inoculated to Xanthi nc, the numberof local lesions in inoculated leaves was counted three days from theinoculation and was compared to lesions of non-transgenic individuals(control). FIG. 1 shows the appearance of an inoculated leaf fromtransgenic Xanthi nc tobacco (at the left) and an inoculated leaf fromnon-transgenic Xanthi nc tobacco (at the right).

When cucumber mosaic virus N-strain (CMV-N) was inoculated to Xanthi nc,the number of local lesions in inoculated leaves was counted four daysfrom the inoculation and was compared to lesions of the control. Theresults are shown in Table 3.

                  TABLE 3    ______________________________________    Resistance of Transgenic Plants to CMV-N           Number of Lesions             Inoculation to a leaf immedi-                              Inoculation to the    Strain   ately above the largest leaf                              largest leaf    ______________________________________    SHX-31    71 (108)        48 (79)    SHX-32   17 (26)          18 (30)    SHX-34   14 (21)           9 (15)    SHX-35   19 (29)           6 (10)    SHX-36   14 (21)          13 (21)    SHX-37   20 (30)          15 (25)    SHX-38    7 (11)          10 (16)    SHX-33   13 (20)          11 (18)              7 (l1)          5 (8)    SHX-39    5 (18)          4 (7)              8 (12)           6 (10)    SHX-40   16 (24)          13 (21)             16 (24)          10 (16)    Control  66               61    ______________________________________

The inoculation of CMV-N was carried out on two different leaves, i.e.,the largest leaf and a leaf immediately above the largest leaf.

With respect to the seven strains of SHX-31, SHX-32, SHX-34, SHX-35,SHX-36, SHX-37 and SHX-38, only one clone individual for each wassubjected to the experiment. With respect to the three strains ofSHX-33, SHX-39 and SHX-40, two clone individuals for each were subjectedto the experiment. With respect to the non-transgenic plant, the numberof lesions in five individuals were counted and the mean value wascalculated. The figures in parentheses represent the percentage of thenumber of lesions observed in each strain based on the number of lesionsobserved in the non-transgenic plant. The difference in values betweenthe transgenic individuals and the non-transgenic individuals wassignificant in the analysis of variance (ANOVA).

When potato virus Y (PVY) was inoculated to BY-4 and Xanthi nc, theamount of viral growth in upper leaves was assayed by ELISA about twoweeks from the inoculation. The results are shown in Table 4.

                  TABLE 4    ______________________________________    Resistance of Transgenic Plants to PVY    Strain         Morphology PVY Concentration    (R.sub.0)      in Appearance                              (μg/g fresh weight)    ______________________________________    Control                   4.2                              3.7    Individuals developing    symptoms (7/10)    SHB18          SS         3.9    SHB19          SS         2.8    Individuals developing    no symptoms (3/10)    SHB4           SS, G, R   2.1    SHB13          SS, G, R   1.2    SHB14          SS, G, R   1.4    ______________________________________     SS: lateral buds, G: delayed aging, R: dwarfing

The values in the Table represent amounts of PVY in plant tissuedetermined by ELIZA. The difference in value was significant in theanalysis of variance (ANOVA).

From the results so far described, the SAHH transgenic organism has beenfound to be resistant to TMV, CMV and PVY which are the three majorviruses causing diseases in tobacco.

EXAMPLE 4! Inhibition of SAHH Gene in Yeast

From the above-described Examples, it has been confirmed that variouschanges are induced in plants by integrating SAHH gene into the plants'genomic genes in such a manner that the gene is expressed in theantisense direction. However, all of those changes described in aboveExamples are observed at the individual level, i.e., in multicellularsystems. Accordingly, there is a possibility that those changes mighthave been induced by some factor(s) other than the introduction of theantisense gene. Then, in order to determine whether the expressioninhibition of SAHH gene is occurring at the cellular level, thefollowing experiment was conducted using Tyl transposon.

Since Tyl transposon of yeast performs replication and particleformation which extremely resemble those of animal retroviruses, modelexperiments for the study of retroviruses or research and development ofantiviral agents (Natsoulis et al., Natung this system (Natsoulis etal., Nature 352, 632).

Propagated Tyl integrates its own sequence into the nuclear DNA of yeast(transposition). When SAHH gene which will be expressed in the antisensedirection has been transferred into the yeast in advance, the synthesisof SAHH will be inhibited and, as a result, the frequency of Tyltransposition is thought to decline corresponding to the degree of theabove inhibition. Therefore, by examining the frequency oftransposition, it is possible to know the degree of the expressioninhibition of SAHH gene. The frequency of transposition can be known byintroducing into yeast a plasmid containing a Tyl clone and aneomycin-resistant marker gene, culturing the yeast and transferring theyeast to a neomycin-containing medium.

Hereinbelow, the specific procedures of the subject experiment will bedescribed. The subject experiment was conducted according to the methoddescribed in "Yeast: A Practical Approach" (Campell et al. (eds.), 1988,IRL PRESS).

Yeast YPH499(a) (Stratagene) was suspended in water to give a celldensity of 6×10¹⁰. Fifty microliters of this suspension was placed in acuvette with a gap of 0.2 cm and mixed with plasmid pJEF1678 containinga clone of the retrotransposon Tyl and a G418-resistant gene. Thisplasmid pJEF1678 was released from Dr. Boeke in the United States. Theyeast suspension mixed with the plasmid was placed in a chamber of anelectroporator (Gene Pulser manufactured by Bio Rad) and a pulse wasapplied once at 0.6 kV and 25 μF, to thereby introduce plasmid pJEF1678into yeast YPH499(a).

Subsequently, tobacco SAHH gene was subcloned into yeast plasmid pYEUra3(Clonetech) at the BamHI-XhoI site so that the gene would be transcribedfrom gall promoter and expressed in the antisense direction(pYEUra3-TobSAHH). This plasmid was introduced into yeast YPH500 (α)(Stratagene) in a similar manner to that described in the introductionof pJEF1678 into yeast.

The two yeast strains into which a plasmid had been thus introduced weremated in a synthetic minimal galactose medium lacking histidine anduracil. While allowing the two plasmids to coexist, the two strains werecultured at 30° C. for two days. Then, mated yeast cells were culturedin a synthetic minimal glucose medium lacking histidine and uracil at22° C. for 5-7 days to form colonies. At this time, transposition isinduced. Colonies thus formed were transferred to a synthetic minimalglucose medium lacking histidine and uracil, cultured at 30° C. for twodays and further cultured in a synthetic liquid glucose medium onlylacking uracil at 30° C. overnight. By these treatments,pJEF1678-eliminated cells are generated. The resultant culture broth wasdiluted with water and seeded in a solid glucose medium only lackinguracil. Cells were cultured at 30° C. for two days. After thiscultivation, the yeast was streaked on a synthetic minimal glucosemedium only lacking uracil and on a synthetic minimal glucose mediumlacking histidine and uracil. By selecting those colonies which did notgrow in the latter medium but grew in the former medium, there wasselected only the yeast from which plasmid pJEF1678 had been eliminated.The reason why only the plasmid-eliminated yeast was selected here isthat resistance to neomycin should be derived from transposition, notfrom the plasmid.

The yeast selected by the above procedures was streaked on auracil-lacking glucose medium supplemented with 500 μg/ml of theantibiotic G418. Then, the numbers of G418 resistant colonies andsensitive colonies were counted. As control, yeast YPH499(a) into whichpYEUra3 had been introduced instead of pYEUra3-TobSAHH was cultured in amedium containing aristelomycin and the numbers of G418 resistantcolonies and sensitive colonies were counted similarly as describedabove. The results are shown in Table 5.

                  TABLE 5    ______________________________________    Effects of SAHH Inhibition upon Tyl Transposon    ______________________________________    Aristelomycin                No. of Resistant                            No. of Sensitive                                        Inhibition    Concentration                Colonies    Colonies    Ratio    ______________________________________     0 (μg/ml)                73           7          NA    100         66          14          10    500         50          30          32    ______________________________________    Plasmid     No. of Resistant                            No. of Sensitive                                        Inhibition    Introduced  Colonies    Colonies    Ratio    ______________________________________    pYEUra3     74           6          NA    pYEUra3-TobSAHH                66          14          12    ______________________________________

As Table 5 shows, when pYEUra3-TobSAHH was introduced into yeast, anincrease in the number of sensitive colonies was observed compared tocontrol. This means that the introduction of the antisense SAHH geneinhibited the transposition of Tyl transposon into the nuclear DNA.Thus, the fact that the inhibition of expression of SAHH gene hasoccurred in cells Per se suggests that the SAHH inhibition of thepresent invention is possible not only in plants illustrated in Examplesbut also in other organisms such as fungi and animals.

EXAMPLE 5! Effects of SAHH upon Endogenous Cytokinins

The expression of antisense SAHH RNA and a reduction in thecorresponding sense RNA associated with the expression were analyzed byNorthern blotting. Briefly, mRNA was extracted from tobacco leaves andseparated with agarose gel. Then, RNA was transferred to a nylonmembrane. Subsequently, SAHH mRNA (sense) or antisense RNA wassynthesized in vitro. Using these riboprobes, hybridization wasperformed. The results are shown in FIGS. 6A. and 6B in the Figurerepresents non-transgenic BY-4 and "X" represents non-transgenic Xanthinc. Further, "C" represents mRNA extracted from a flower head portion ofthe control plant. As the Figure shows, the antisense RNA is synthesizedin transgenic plants and a reduction in the sense RNA is observed there.

In addition, the amount of endogenous cytokinins in transgenic tobaccowas also determined at this time. Briefly, root exudate of tobacco wasrecovered and medium components were added thereto. Then, a bioassay wasconducted on tobacco callus. "A" represents the cytokinin content intransgenic Xanthi nc tobacco and "B" the cytokinin content innon-transgenic Xanthi nc tobacco. "C" represents the cytokinin contentwhen anti-cytokinin was added to the root exudate to thereby suppressthe activity of cytokinin. As the Figure shows, the cytokinin content(A) in transgenic tobacco has increased almost three times compared tothe cytokinin content (B) in non-transgenic tobacco.

Considering the above results shown in FIGS. 6A-6B and 7, it is presumedthat SAHH was reduced by the antisense inhibition of SAHH and as aresult, endogeneous cytokinins have been increased.

Effect of the Invention

The present invention provides those organisms in which the expressionof SAHH gene is substantially inhibited. Such organisms have excellentproperties, such as resistance to viruses, and are industrially useful.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 4    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1812 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA to mRNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    - GAAGAGAAAA AAGCCTCTCA AATCTCATCT CTAACCACCC AATTTCTCAT AC - #TCGCTCTA      60    - CCCATGGCTC TATTAGTCGA GAAGACCACC TCTGGCCGCG AGTACAAGGT CA - #AGGACATG     120    - TCTCAGGCCG ATTTCGGCCG GCTTGAAATC GAGCTGGCCG AAGTTGAAAT GC - #CTGGTCTC     180    - ATGGCTTGTC GTACTGAATT TGGCCCTTCA CAGCCATTTA AAGGTGCTAA GA - #TTACTGGA     240    - TCTTTACATA TGACCATTCA AACTGCAGTT TTGATTGAAA CCCTTACTGC TT - #TGGGTGCT     300    - GAAGTTAGAT GGTGTTCTTG CAACATCTTC TCCACTCAAG ATCACGCCGC TG - #CTGCCATT     360    - GCACGTGACA GCGCCGCCGT GTTCGCGTGG AAGGGTGAGA CTCTGCAGGA GT - #ATTGGTGG     420    - TGTACTGAGA GGGCACTTGA CTGGGGTCCA GGTGGTGGGC CCGACTTGAT CG - #TCGACGAT     480    - GGTGGTGATG CTACACTCTT GATTCATGAG GGTGTTAAGG CAGAAGAAGA GT - #TTGCTAAG     540    - AATGGGACAA TCCCAGATCC TAACTCTACC GATAATGCTG AGTTTCAGCT TG - #TACTTACT     600    - ATTATTAAGG AAAGTTTGAA GACTGATCCT TTAAAATATA CCAAGATGAA GG - #AAAGACTC     660    - GTCGGTGTTT CTGAGGAAAC TACCACTGGA GTTAAGAGGC TTTATCAGAT GC - #AGGCTAAT     720    - GGAACTTTGC TTTTCCCTGC TATTAATGTT AATGATTCTG TTACCAAGAG CA - #AGTTCGAC     780    - AACTTGTACG GATGCCGCCA CTCACTGCCC GATGGTCTCA TGAGGGCTAC TG - #ATGTTATG     840    - ATTGCCGGAA AGGTTGCCCT TGTTGCTGGT TATGGAGATG TCGGCAAGGG TT - #GTGCTGCT     900    - GCCTTGAAAC AAGCCGGTGC CCGTGTGATT GTGACCGAGA TTGACCCTAT CT - #GTGCTCTC     960    - CAGGCTACCA TGGAAGGCCT CCAGGTCCTT ACTCTAGAGG ATGTCGTTTC TG - #ATGTTGAT    1020    - ATCTTTGTCA CCACGACCGG TAACAAGGAC ATTATCATGG TTGACCACAT GA - #GGAAGATG    1080    - AAGAACAATG CCATTGTTTG CAACATTGGT CACTTTGACA ACGAAATCGA CA - #TGCTTGGT    1140    - CTCGAGACCT ACCCTGGTGT CAAGAGGATC ACAATTAAGC CTCAAACCGA CA - #GATGGGTC    1200    - TTCCCTGACA CCAACAGTGG CATCATTGTC TTGGCTGAGG GTCGTCTCAT GA - #ACTTGGGA    1260    - TGTGCCACAG GACACCCTAG TTTTGTGATG TCGTGCTCGT TCACTAACCA AG - #TCATTGCC    1320    - CAACTCGAGT TGTGGAATGA AAAGAGCAGT GGGAAGTATG AGAAGAAAGT GT - #ATGTCTTG    1380    - CCAAAACACC TCGACGAGAA GGTTGCTGCA CTTCATCTCG GAAAGCTCGG AG - #CCAAGCTT    1440    - ACCAAACTTT CGAAGGATCA AGCTGACTAC ATTAGCGTTC CAGTTGAGGG TC - #CTTACAAG    1500    - CCTGCTCACT ACAGGTACTG AGCGAAAACA AATCGACAGA GGAGAACAGC AT - #TGTCGCGG    1560    - CATGATTGTT TTGCATTTAA TACTTTGATT TTGTTTAGGA TACTAGTATT TT - #GAATATTG    1620    - GTGGTGATAT ATTTGGGAGG AAGTGGCATG TTTTGCTGGA AAAGAAATGG GT - #CTTATTTG    1680    - AAAGTAAGAC CAAAATGTGT TGAATAAGAT TATGGTTGGT GGTGTGATAT GA - #TATTGTAG    1740    - TAAGTTAGAA CCATTTGCTT TTTGGTGTAT GGTTTTTGTT TCAAGAAATC AA - #AGCAACAC    1800    #     1812    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 34 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: other nucleic acid    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    #        34        NNNN GGACGAAACG GUCU    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: other nucleic acid    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    # 20               CGTC    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: other nucleic acid    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    # 20               GCCG    __________________________________________________________________________

What is claimed is:
 1. A transgenic plant in which the expression ofS-adenosylhomocysteine hydrolase (SAHH) gene present in its genome isinhibited by inserting all or part of an SAHH gene into the genomic DNAof said plant in a reverse direction such that the reversed SAHH geneinduces transcription of an antisense RNA against the endogenous SAHHgene and SAHH enzymatic activity is inhibited.
 2. A method for creatinga transgenic plant in which the expression of SAHH is inhibited,comprising integrating into the genomic DNA of said plant all or part ofan SAHH gene in a reverse direction, such that the reversed SAHH geneinduces transcription of an antisense RNA against the endogenous SAHHgene and SAHH enzymatic activity is inhibited.
 3. The transgenic plantof claim 1, wherein said plant is a dicotyledon or a monocotyledon. 4.The transgenic plant of claim 1, wherein said plant is an angiosperm ora gymnosperm.
 5. The transgenic plant of claim 3, wherein saidmonocotyledon or dicotyledon is selected from the group consisting oftobacco, tomato, potato, rose, lily, maize and rice.
 6. A method ofincreasing cytokinin concentrations in a plant comprising inhibitingexpression of an SAHH gene present in the plant by integrating into thegenome of said plant all or part of an SAHH gene inserted in a reversedirection, such that the reversed SAHH gene induces transcription of anantisense RNA against the endogenous SAHH gene and SAHH enzymaticactivity is inhibited.